7-AAD Red Fluorescent Live/Dead Stain

1. Add 1 part Antigen-Down HRP Conjugate Stabilizer to 4 parts deionized water and mix for 5-15 minutes., 2. Add the anti-IgG-HRP conjugate directly into the conjugate diluent to a working 1X dilution and mix for 5-15 minutes., 3. Pipette diluted conjugate onto the plate, or store protected from light at 2-8°C for use at a later date. 1X working conjugate dilutions can be stored at 2-8°C and protected from light, providing a useful shelf-life of 6-12 months.

7-Aminoactinomycin D (7-AAD, catalog 163) is an intercalating red fluorescent reagent that binds between cytosine and guanine bases of DNA in membrane-compromised cells. This material, like its parent molecule, Actinomycin D, is a DNA-intercalator with growth-inhibitory properties. Normal healthy cells, with intact membrane structure, will exclude the polar 7-AAD vital dye. Mid to late apoptotic and necrotic cells will not exclude this dye and subsequently stain red when 7-AAD complexes with the nuclear DNA. As 7-AAD is membrane impermeant, it cannot reach the DNA in viable cells, thus allowing the identification of cells with permeabilized membranes in a population. 7-AAD distinguishes between living and dead cells by counterstaining nucleic acids red in necrotic, dead, dying, and membrane-compromised cells, while the DNA in healthy cells remains unstained. 7-AAD is supplied as a highly concentrated lyophilized powder at 0.26 mg/vial. Reconstitute it with 260 µL DMSO to yield a stock concentrate at 1 mg/mL. Add it to the cells at a final concentration of 5 µg/mL and analyze with a flow cytometer (Figure 1) or fluorescence microscope (Figure 2). When bound to nucleic acids, the maximum absorption is 546 nm and the maximum emission is 647 nm. 7-aminoactinomycin D may be used in combination with other fluorescent reagents for dual staining or multiplexing purposes. Use it with our FAM-FLICA® poly caspases inhibitor reagent (catalog 637) to include apoptotic cells in the analysis. When used together, these reagents will identify four populations of cells: living; early apoptotic; late apoptotic; and necrotic cells leading to more accurate results when assessing cell death (Figures 2 and 3).